University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 12-2008 Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation Blake Lee Joyce University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Part of the Plant Sciences Commons Recommended Citation Joyce, Blake Lee, "Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation. " Master's Thesis, University of Tennessee, 2008. https://trace.tennessee.edu/utk_gradthes/452 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Blake Lee Joyce entitled "Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Plant Sciences. C. Neal Stewart, Major Professor We have read this thesis and recommend its acceptance: Beth Mullin, Zong-Ming Cheng, Arnold Saxton Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a thesis written by Blake Lee Joyce entitled “Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation.” I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Plant Science. ________________________ C. Neal Stewart, Jr. Major Professor We have read this thesis and recommend its acceptance: _____________________________ Beth Mullin _____________________________ Zong-Ming Cheng _____________________________ Arnold Saxton Accepted for the Council: _____________________________ Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Characterization of the Metallohistin cDNA AgNt84 and Pteris vittata Tissue Culture for Phytoremediation A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Blake Lee Joyce December 2008 Acknowledgements First, I wish to thank Neal Stewart for the support, funding, and mentoring that he has provided me. Above all, I am most thankful for his kindness and constant belief in me. Secondly, I wish to thank Beth Mullin for the seemingly infinite amount of patience and time she has spent with me throughout this project. I also want to thank my committee members Arnold Saxton and Zong-Ming Chang for all of their assistance in improving both this thesis and my understanding of the scientific process. I owe a great debt of gratitude to all the people in the Stewart lab. I would like to specially thank Jason Abercrombie for mentoring me throughout my project. I am also very grateful to Mitra Mazarei, Murali Rao, Dave Mann, Hani Al-Ahmad, Laura Good Abercrombie, Wusheng Liu, Brian Leckie, Reginald Millwood, and Jason Burris for all of their assistance. To all of my family and friends, I want thank you for your eternal enthusiasm, support, understanding and love. It is for you and the hope of improving your world that I originally began in the scientific field. Lastly, I want to thank Bridgid Lammers for helping to make a home for me in Knoxville, TN. ii Abstract Contamination of soils with toxic metals such as arsenic and cadmium has become a major environmental and human health risk. Phytoremediation provides a method to remove contaminants from soils that is not only economically viable but also environmentally sound. Metallohistins are proteins that have the capability to bind divalent metal ions such as Ni2+, Zn2+, Co2+, Cu2+ and Cd2+. In this study, a concatemer sequence was designed to try to increase the presence of metal-binding proteins in transgenic plants. Two methods to increase translational efficiency of the metallohistin protein were used: 1) characterization of the full-length metallohistin AgNt84 gene, and 2) construction of three vectors containing different fragments of the AgNt84 cDNA which were transformed into Nicotiana tabacum. The concatemer sequence proved toxic to Escherichia coli cells and could not be cloned into vectors for plant transformation. Explants genetically transformed with vectors containing either the entire AgNt84 cDNA or the 5’ untranslated and coding region of the cDNA recovered from tissue culture. Explants genetically transformed with a vector containing only the coding region of the cDNA produced shoots but not roots in tissue culture, and then became necrotic. Characterization of the transformants is underway. The first exon and portion of the intron of the gene has been sequenced. Phytosensors that can recognize and report the presence of arsenic would provide remediators with a management tool for phytoremediation. A transmission and scanning electron microscopy study of Pteris vittata tissue culture revealed callus formation on iii epidermal cells of gametophytes, presence of an extracellular matrix on calli, and the formation of croziers during differentiation. Calli induced on semi-solid medium consisted of distinct meristematic nodules. These nodules differentiated randomly, and are unfit for genetic transformation. A new differentiation medium is also described. A preliminary genetic transformation study was successful in creating protoplasts from both Pteris vittata gametophytes and sporophytes, but unsuccessful with biolistic bombardment of calli. Low yields, cellular debris, and autofluorescence exhibited by the protoplasts hampered polyethylene glycol-mediated genetic transformation and detection of transgene expression. iv Table of Contents Page Chapter One: Introduction ................................................................................................. 1 Remediation of contaminated soil .................................................................................. 1 Metal toxicity .................................................................................................................. 5 Metallohistins .................................................................................................................. 7 Hyperaccumulators, bioremediation, and heavy metals ................................................. 9 The use of Pteris vittata for phytoremediation .............................................................. 12 Tissue culture of Pteris vittata .................................................................................. 13 Possible transformation techniques........................................................................... 15 Fern genomics, transcriptomics, and proteomics ...................................................... 17 Objectives ..................................................................................................................... 20 Chapter Two: Assessing the Phytoremediation Potential of the Metallohistin cDNA AgNt84 .............................................................................................................................. 21 Introduction ................................................................................................................... 21 Methods and Materials .................................................................................................. 24 cDNA constructs ....................................................................................................... 24 Agrobacterium-mediated transformation of Nicotiana tabacum .............................. 26 v Full length gene discovery ........................................................................................ 27 Results ........................................................................................................................... 28 Concatemer sequence................................................................................................ 28 cDNA constructs and transformation of Nicotiana tabacum .................................... 29 AgNt84 gene discovery ............................................................................................. 31 Discussion ..................................................................................................................... 34 Chapter Three: SEM and TEM Characterization of Pteris vittata Callus Induction and Regeneration ..................................................................................................................... 42 Introduction ................................................................................................................... 42 Materials and Methods .................................................................................................. 46 Environmental conditions and tissue culture ............................................................ 46 Fixation ....................................................................................................................